Naturalkiller Cell Biology Natural killer (NK) cells were originally described in termsof function in 1971 when Cudkowizc and Bennett observed that mice that hadreceived lethal irradiation were capable of rejecting allogeneic or parentalstrain bone marrow cell (BMC) allografts.(1, 2) NK cells are of lymphoid originand are found in the peripheral blood, (constituteapproximately 10% of the lymphocytes in human peripheral blood), spleen, and BM, aswell as other tissues. They are radio-resistant, large, granularlymphocytes that representan important arm of innate immunity and are thought to play a critical role inthe immune surveillance against tumors and virally infected cells.

(3)They areregulated by a number of receptors with opposite function that finely tunepotent effector functions such as cytolytic activity and production ofcytokines playing a major role in inflammation and regulation of both innateand adaptive immune responses(4-9) NK cells cankill in a rapid manner and this NK cell-mediated cytotoxicity occurs primarilythrough the perforin/granzyme-dependent pathway, although NK cells can also useFas ligand (FasL) and tumor necrosis factor related apoptosis inducing ligand(TRAIL) to kill target cells (10). NK cells also have the ability to secrete a widerange of cytokines upon activation (11) and these cytokines can either promoteor inhibit hematopoiesis, for example, GM-CSF and TGF-?, respectively(10-12).Cytokinesalso play a key role in the differentiation of NK cells. Interleukin (IL)-2,IL-15, and IL-21, are capable of inducing proliferation and activation of NKcells. However, only IL-15 and fms-like tyrosine kinase 3 (flt3) ligand havebeen shown to be critical for NK cell development and maintenance (13-18). Stemcell factor (SCF) and fetal liver kinase ligand (flk2L) have also been shown tobe important in NK cell differentiation (19)Thefunction of NK cells is also mediated by cytokines.IL-12 and IFN-?/? exertpotent stimulatory effects on NK cells, and IL-12 and IL-18 in combination isparticularly effective in augmenting NK cell function (20, 21).

IL-2 has alsobeen shown to significantly activate NK cells, and adoptive immunotherapy ofIL-2 activated NK cells after autologous BMT has been used in patients with cancerwith acceptable toxicities.(22) The predominant cytolytic targets of NK cells are uncommon cellsthat have downregulated expression of class I MHC(MHC-I), which is expressed onnearly every healthy cell of the body.(23) MHC-Iloss is a fairly common mechanism by which tumors and virus-infected cells canevade recognition by the T-cell receptor of cytolytic T cells, and NK cells.(24,25)Recently, NK cells were officially classified as theprototypical members of the group 1 innate lymphoid cells (ILCs), which aredefined by their capacity to secrete IFN-? but not type 2 cytokines (IL-4 andIL-13), IL-17, or IL-22. Group 1 ILCs are thus distinct from group 2 ILCs,which produce IL-13, and group 3 ILCs, which can produce IL-17, IL-22, or both.(26,27)Human NK cells are classically defined as CD56+CD3- cells, distinguishing them from CD56+CD3+ cells, which consist of a mixed population ofNK-like T cells and antigen-experienced T cells that have upregulated severalNK cell markers.(28)Two major subsetsof NK cells are found in human subjects that can be distinguished by theirlevels of CD56 expression, namely CD56dim and CD56bright.

(28) CD56dimNK cells are fully mature, make up approximately 90% of the NK cells inperipheral blood, and predominantly mediate cytotoxicity responses. Incontrast, CD56bright cells are more immature, make up approximately5% to 15% of total NK cells, and have been considered primarily as cytokineproducers while playing a limited role in cytolytic responses. Although CD56brightNK cells are more efficient at producing cytokines overall, the CD56dimNK cells can also contribute significantly to early cytokine production becausethey comprise a significantly greater fraction of the total NK cell pool andcan more rapidly secrete cytokines.(29-31)NK CellReceptorsTheidentification of MHC class I-specific inhibitory and activating receptors inaddition to non-MHC class I specific activating and inhibitory receptors hasgrown substantially in the two past decades. This has led to a tremendously complexset of receptors responsible for innate recognition of foreign, abnormal, orvirally infected cells by NK cells, and these receptors have become relevantwith respect to allogeneic BMT and malignant cancer therapies.(1) The firstclass of MHC class I-specific receptors was identified in mouse and is termedLy49.

In any given mouse, an NK cell population can express a variablecombination of the 20 or more Ly49 receptors characterized. Some of thesereceptors have different affinities for the different MHC class Imolecules.(2,3)The nextclass of receptors are the killer cell Ig-like receptors (KIRs) whichspecifically recognize groups of HLA-C (p58 or KIR2DL1-KIR3DL3)(4-7), HLA-B(p70 or KIR3DL1) and HLA-A alleles (p140) (8-13) Both the Ly49 and KIRmulti-receptor families contain members that have either activating orinhibitory action.

(14)Another classof MHC class I-specific receptors expressed both in humans and in mice iscomprised of the C-type lectin molecule CD94, which is covalently associatedwith a member of the NKG2 family. Like Ly49 and KIR receptors, these receptorsalso have been shown to exert inhibitory (NKG2A or NKG2B) or activating (NKG2C)signals upon binding Qa-1b (mouse) or non-classical HLA-E (human) molecules ontargets (15-18).NK cells alsohave Ig-like transcript (ILT) receptors that interact with HLA-G (to protectthe fetus and placenta from rejection)(19). Another group of NK cell receptorscomes from a more diverse family of receptors of NK-cell-specific Ig-likemolecules that are known as natural cytotoxicity receptors, or NCRs. NCRsinclude NKp30, NKp46, and NKp44 as well as NKG2D. NKG2D is a member of the NKG2family expressed by NK cells and cytotoxic lymphocytes (CTLs) (20-23). most NKcells can express the Fc?RIII (CD16) molecule, which recognizes the Fccomponent of bound Ig molecules and initiates cytolysis by the antibodydependent cellular cytotoxicity (ADCC) pathway (24), thus giving the NK cellanother method of target recognition.

(1)Finally,natural killer cells have 5 main categories of cell surface receptors. Activating  receptors (e.g. CD16, NKp46, NKG2D, NKG2C,KIR-S, Ly9), Inhibitory receptors (e.g. KIR-L, NKG2A), chemotactic receptors(e.g. CCR2,CCR5,CXCR1,CXCR4,CXCR6), Cytokine receptors(e.

g.  IL-1R, IL-15R,IL-18R), Adhesionreceptors(e.g. ?1 integrins).

Nk & GVHDAllogeneic bone marrowtransplantation (BMT) has proven to be an effective treatment for hematologicmalignancies and some solid tumors.(1) However, the high incidence ofgraft-versus-host disease (GVHD) as a complication of this treatment haslimited the overall effectiveness of BMT.(2) GVHD is mediated by the activationand proliferation of alloreactive T cells leading to tissue damage in the host,primarily in the gastrointestinal tract, liver, and skin (3) causingsignificant morbidity and mortality.One of the major challenges ofallo-Stem Cell Transplantation(SCT) is to reduce the incidence and severity ofGVHD while boosting the graft-versus-leukemia (GVL) effect.

In the setting ofallo-SCT, the reconstitution of natural killer (NK) cells is of notableinterest due to their known capability to induce GVL without GVHD.(4) Studies of the role of NK cells in bone marrow engraftmentdemonstrated that host NK cells persisting after conditioning can contribute tograft rejection (5) while donor NK cells can promote hematopoieticengraftment (6).The first study suggesting arelationship between NK cells and GvHD development was reported by Lopez andcoworkers from the Sloan Kettering Cancer Center showing a significantassociation between GvHD development and pre-transplant levels of NK cellactivity, as measured by cytotoxic assays performed using herpes simplex virustype 1-infected fibroblast as target cells, in peripheral blood of a small andheterogeneous cohort of 13 patients undergoing different protocols of HCT.(7) Livnat et al. (8) and Dokhelar et al. (9) addressed the sameissue assessing NK cell activity against the K562 leukemic cell line bothbefore and after hematopoietic cell transplantation (HCT) and obtained contradictory results finding either norelationship (8) or a positive association (9) between early post transplant NKcell activity and GvHD development. In 2015Jacobs B.

and et al could demonstrate that NK cells gain cytotoxic andcytokine producing functions early during hematopoietic immune reconstitutionfollowing autologous SCT.(10) In addition to clinical studies, it has beenshown in animal models that IL-2-activated NK cells may efficiently prevent oreven reduce GVHD without any adverse impact on their important GVL effect.(11-13)After chemotherapy or hematopoieticstem cell transplantation, NK cells are the first lymphoid cells to recover (14,15).Surprisingly, such postgrafting regeneration of NK cells does not causeclinical graft-versus-host disease (GVHD); this has led to the conclusion thatnormal nonhematopoietic tissues lack ligands able to activate NK cell lysis.(16)The concept of an NK-mediatedregulatory function is also supported by the observation that a higher numberof bone marrow NK cells has been associated with a decreased incidence ofchronic GVHD after HLA identical sibling bone marrow transplants in human.

(17)Researchesin 2002 and 2004 showed that this regulatory function can be indirect, throughthe interplay and molecular crosstalk with dendritic cells (DCs) (18,19). Onthe one hand, DCs can prime, further the activation of, augment the expansionof, and enhance the activities of NK cells through the production of cytokinessuch as IL-2, IL-12, IL-15, IFN-a/b, and TNFa.(16) The regulatory function ofNK cells on adaptive immuneresponses appears also to be mediated through directlysis of activated T cells (20,21). This pathway has been postulated to play animportant role in the generation of memory T cell repertoire. Several recentobservations suggest that certain subpopulations of NK cells promote allografttolerance via a cytolysis-dependent regulatory pathway (22–24). However, littleis known about the effects of NK cells on donor T cells after BMT. In 2010,Magali Noval Rivas et al showed that NK cells can regulate chronic GVHD bylimiting recipient minor histocompatibility Ag (mHA)-driven proliferation ofdonor CD4+ T cells.

(16)According to studies by scientists, the relationship between NK Cell and GVHD can be generally described in two ways: firstly, Nk Cell cytotoxic functions and GvHD prevention: NK cells can suppress GvHD development through their cytotoxic function either directly, by depleting activated alloreactive T cells, or indirectly, by depleting APC and preventing T cell stimulation,T cell killing by NK cells appears to be dependent on both perforin production (25,26,27) and FAS-mediated induction of apoptosis (26, 28, 29),and secondary Nk Cell cytokine production and GvHD induction: Although it is unclear if NK cells production of immunesuppressivecytokines can prevent GvHD, it isestablished that pro-inflammatory cytokine production by NK cells cancontribute to GvHD development. Xun et al. showed In a xenogeneic model that invitro interleukin-2 (IL-2)-activated human NK cells producing interferon-?(IFN-?)and tumor necrosis factor-?(TNF-?) were able to induce acute GVHD upon transferinto SCID mice (30, 31) What is now important about NK cells and its impact onGVHD is that researchers are looking for new ways to reduce GVHD with the helpof these cells, which we will continue to mention theseactivities.natural Killer Cell MemoryImmunological memory is a cardinalfeature of adaptive immunity.

Although natural killer (NK) cells have long beenconsidered short-lived innate lymphocytes that respond rapidly to transformedand virus-infected cells without prior sensitization.(1) It has recently becomeappreciated that NK cells can also acquire functional qualities commonlyassociated with immunological memory similar to that of T and B cells inresponse to pathogens and in non-infectious settings.(2)Antigen-specific memory NK cell responses were first observed in amurine model of hapten-induced CHS (3). This model was established throughsensitization via painting a specific hapten, such as 2,4-dinitrofluorobenzene(DNFB) or oxazolone (OXA), on mouse skin and subsequent challenge with the samehapten on the ears of the mice, after which the recall responses to the haptenswere measured based on ear swelling.(4,5)The ability of the immune system torespond rapidly and provide enhanced protection of the host against apreviously encountered pathogen is defined classically as immunological memory.Longlived memory cells are generated after initial infection and displayheightened responses upon secondary challenge with the same pathogen.

Theprocess of memory formation in T cells has been well studied and is generallydivided into three distinct phases. Upon exposure to cognate antigen, naiveCD8+ T cells clonally expand and differentiate into effector cells during the”expansion” phase. This first phase is followed by a rapid ”contraction”phase, when the vast majority of effector CD8+ T cells undergo apoptosis toform a small, but stable, pool of surviving cells that then enter the third”memory” phase. Memory CD8+ T cells persist throughout the host organs andmaintain their longevity through self-renewal until a subsequent encounter withtheir cognate antigen, when they exhibit enhanced effector function and hostprotection. In an experimental system in which Ly49H+ NK cells were adoptivelytransferred into mice lacking this receptor, these Ly49H+ cells underwentrobust antigen-driven expansion after MCMV infection.

Similar to activated CD4+T cells, expanded effector NK cells undergo a slower and sustained contractionphase to establish a long-lived and self-renewing ”memory” pool ofantigen-specific NK cells that can be recovered many months after infection ina variety of peripheral tissues. (6)In the following section, wesummarized the receptors, cytokines and signalling pathways that have been impli­catedin the development of memory NK cells:1.      Hapten-specific memory NK cells:Following sensitization of mice with haptens, hapten-specific memory naturalkiller (NK) cells are detected in the liver. The generation of these memory NKcells is dependent on the cytokines interleukin?12 (IL?12), interferon?? (IFN?)and IFN?. Antibody-mediated blockade of the NK cell receptor natural killergroup 2, member D (NKG2D) or CXC-chemokine receptor 6 (CXCR6), or moleculesthat are involved in NK cell trafficking (such as CD18 and P- and E?selectin)prevents the development of contact hypersensitivity (CHS) responses in the earafter hapten challenge.(3,7,8)2.      MCMV-specific memory NK cells:During mouse cytomegalovirus (MCMV)-infection, naive NK cells expressing theLY49H receptor expand with contribution of signalling mediated by the DNAXaccessory molecule 1 (DNAM1) receptor and the inflammatory cytokines IL?12, IL?18and IL?33. Inflammatory cytokines drive the expression of zinc finger and BTBdomain-containing 32 (ZBTB32) and the microRNA miR?155, which are involved inthe expansion of ‘effector’ NK cells.

Following the elimination of the virus,the BIM and autophagy pathways regulate the contraction of the expandedpopulations of NK cells, giving rise to a population of MCMV-specific memory NKcells. Although MCMV-specific memory NK cells distribute systemically in mice,memory NK cells specific for vaccinia virus reside in the liver, and influenzavirus-specific memory NK cells are found in the liver and lungs (not shown). Innon-human primates, simian immunodeficiency virus-specific memory NK cellsreside in the spleen and liver.

(9,10,11,12,13,14)3.      Cytokine-induced memory NK cells: In vitro, thebrief exposure of NK cells to the cytokines IL?12, IL?15 and IL?18 results inthe upregulation of IFN?, perforin and granzymes, and the production of highlevels of CD25, the high-affinity ?-chain of the IL?2 receptor, is alsoinduced. After adoptive transfer, these cytokine-induced memory NK cellspersist long term, and their ability to produce abundant cytokines and expressperforin and granzymes is maintained. The presence of IL?2 (or IL?15) furtherincreases NK cell numbers and their ability to express IFN?, perforin and granzymesafter adoptive transfer.(15,16,17,18)   

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